Cholangiocarcinoma (CC) is an aggressive cancer that has been refractory to most chemotherapeutic or targeted agents. Our goal is to extend success of immunotherapy to CC, and identify relevant biomarkers for this approach. This proposal will translate pre-clinical findings showing that MEK inhibitors (MEKi) enhance infiltration of CD8+ T effector cells, prevent their exhaustive apoptotic cell death, and enhance efficacy of anti-PD-L1 Ab in tumor models. We hypothesize that MEKi will synergize with PD-L1 blockade to elicit anti-tumor responses via altering cytokine and chemokine signatures that promote CD8+ T cell infiltration and survival and decreased immunosuppressive cells in the tumor microenvironment (TME). We will conduct an innovative randomized phase 2 clinical trial to examine safety and efficacy of a MEKi (cobimetinib) in combination with an inhibitor of PD-L1 (atezolizumab) and atezolizumab monotherapy in patients with unresectable CC. This trial will be performed in the context of our approved NCI-sponsored UM1 clinical trials grant. Concurrent studies will elucidate the mechanism by which MEKi acts on T and myeloid cells, and augments efficacy of PD-L1 blockade. The trial will provide paired biopsies from patients with accessible tumor in which we can validate our mechanistic findings using innovative multi-spectral imaging. This will be the largest clinical study of CC in the second line. We will pursue three Specific Aims: 1) To conduct a randomized phase 2 clinical trial of the PD-L1 inhibitor atezolizumab in combination with the MEKi cobimetinib versus atezolizumab monotherapy in participants with unresectable CC. We have completed approval through the NCI Cancer Therapy Evaluation Program (CTEP) to conduct this randomized, multicenter clinical trial in 76 evaluable subjects who have received at least one prior chemotherapy. If successful, this regimen has potential to become the standard of care for CC in the second line. This trial will provide samples to evaluate the effect of MEKi on key biomarkers in the TME. 2) To determine how MEKi alter the TME to improve the response to anti-PD-L1 therapy. A unique panel of human CC cell lines will be used study how MEKi modulates tumor-derived chemokines that regulate T cell migration. Using clinical trial biopsies, we will evaluate CD8+ T cell infiltration and PD-L1 expression as integrated biomarkers. Multi- spectral IHC will characterize the impact of treatment on immune subsets and markers of exhaustive T cell death (i.e. Nur77) in the TME as well as the impact on immunosuppressive T regulatory cells and myeloid-derived suppressor cells. 3) To determine the role of systemic cytokines and immune cell populations in the response to MEKi and PD-L1 blockade. In vivo models will test if the efficacy of MEKi + anti-PD-L1 therapy is dependent on IFN-?, a key cytokine regulator of T cell function and chemokine-mediated trafficking. In vitro studies will define if MEKi alters the impact of tumor-derived cytokines (e.g. IL-6, GM-CSF) on differentiation or function of suppressive myeloid cells. Finally, using patient peripheral blood samples we will determine the relevance of relationships between cytokine expression and various immune cell phenotype with clinical outcome.